JP2006302113A - Electronic medical chart system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electronic medical chart system having a retrieval function based on comparison including measurement data itself to be obtained at an inspection state when a doctor in charge retrieves patients of similar cases showing symptoms and inspection results similar to those of a new patient from cases in the past stored in the electronic medical chart system when the doctor in charge gives diagnosis regarding causes of disease of the new patient. <P>SOLUTION: The electronic medical chart system has a patient electronic medical chart database which stores inspection data, clinical finding data and medical treatment data of a patient, a selection mechanism of the patients showing similar cases for comparing the inspection data, the clinical finding data of the new patient with inspection data and clinical finding data in the past cases in the patient electronic medical chart database to select one or more cases in the past similar to those of the new patient and a medical chart information disclosure mechanism of the patients showing the similar cases for disclosing the contents of the electronic medical charts of the selected patients showing the similar cases to the doctor in charge of the new patient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention is an electronic medical chart system comprising a patient database for electronically storing patient clinical data (treatment data, examination data) and the like, and a search process mechanism for retrieving desired data from the patient database. About. In particular, an electronic medical record system comprising a search process mechanism for retrieving past patient clinical data having similar examination data, etc., in contrast to new patient examination data, etc. from an existing patient database About.

  Medical institutions collect, record, and manage medical information such as patient profiles, medical history, clinical findings, examination data, and medical records as medical records. Previously, medical charts were made on a paper base, but they have been digitized using an electronic filing system. When proceeding with the digitization of this chart, the items to be described and the description form of each item are formulated. For example, in a doctor's examination, a patient's profile, history of onset, information on subjective symptoms, and information on objective symptoms found in the doctor's own observations obtained by an interview are respectively stored in a predetermined manner. The data is recorded on the electronic medical record as a part of clinical finding data after being divided into items and using a certain formulated expression. In addition, when identifying the etiology causing the patient's symptoms, inference is made using the diagnostic knowledge based on the information on the subjective symptoms and the information on the objective symptoms, and possible etiological candidates are selected. Further, various tests are performed in order to identify which etiology is actually among the possible etiology candidates. The various inspection results that have been performed are recorded on the electronic medical chart as inspection data after being classified into predetermined items and using a certain formulated expression. Ultimately, the most probable etiological candidate that may have been inferred in advance using patient profile, history of onset, subjective symptom information, objective symptom information, and various test results Identify high etiology and make a diagnosis. In consideration of the diagnosis (etiology) made and the symptoms of the patient, a suitable treatment is selected from a plurality of treatment means, and treatment is started. Thereafter, a series of treatment records including treatment performed during the treatment period and changes in patient symptoms are recorded on the electronic medical record as treatment data.

In electronic medical records, patient profile, history of onset, information on subjective symptoms, information on objective symptoms, and various test results stored in test data are part of clinical findings data. There has already been proposed a system that uses the advantages stored in a predetermined format and supports the work of selecting a possible etiological candidate by making inferences using diagnostic knowledge at the stage of identification of the etiology by a doctor. (See Patent Document 1). Conventionally, based on information on subjective symptoms and objective symptoms of the patient, inferring using diagnostic knowledge, selecting possible etiological candidates, then considering the test results, When identifying the most likely etiology candidate among certain etiology candidates, most of the abnormal test data associated with the disease included in the test results is consistent with the most likely etiology candidate, Abnormal test data may not be consistent with the most likely etiology candidate. If there is such a discrepancy, it is necessary to reconsider a possible etiology candidate and identify the most likely etiology candidate that matches all of the abnormal test data associated with the disease. The electronic medical record system (Patent Document 1) infers using diagnostic knowledge including information on subjective symptoms of the patient, information on objective symptoms, and test results, and a possible etiological candidate It has a function to support reconsideration and reselection work. That is, the diagnostic support function employed by the electronic medical record system (Patent Document 1) is that, when some abnormal test data does not match the most likely etiology candidate, the doctor overlooks the mismatch. Or, as a result of not paying enough attention, it has the effect of reducing the concern of missing the “true etiology” of the patient.
Japanese Patent Laid-Open No. 10-177605

  Although the conventional electronic medical chart system stores the patient's subjective symptom information, the objective symptom information, and the test result as the chart information of each patient, the stored test result is, for example, blood Based on the information obtained by the test, such as medium cholesterol concentration, blood glucose level, and uric acid level, only the test results that do not require further determination on the medical contents that the test data means. In other words, based on the test result of the healthy person, the test result that can uniquely determine whether or not the abnormal test data exists in the patient test result is stored in accordance with the already established criteria. It was. Therefore, when a doctor diagnoses a patient, based on the patient's symptom and test result, it has a function to support the work of reconsidering and reselecting a possible etiological candidate by inferring using diagnostic knowledge. At that time, the extraction of abnormal test data related to a disease can be inferred with high accuracy using diagnosis knowledge based on typical cases. As long as the test items for which normal or abnormal test criteria have already been established are used to extract the abnormal test data included in the test results, the above-mentioned electronic medical record system diagnosis support function Is valid.

  On the other hand, in addition to patient symptoms and test results that have been used in the past, test data obtained in tests that belong to the state-of-the-art medical field, such as genetic tests, are also used by doctors to identify the etiology of patients. It has become so. However, in an examination belonging to the state-of-the-art medical field, it is often difficult to make a clear interpretation when interpreting the meaning based on the obtained examination data. For example, for a patient suffering from an infectious disease, a probe hybridization method using a DNA microarray comprising DNA probes for detecting a plurality of types of bacteria has been applied to identify the causative bacteria of the infectious disease. . At that time, even if the sample contains causative bacteria belonging to the same species, there is a difference in signal intensity due to the label attached to the nucleic acid molecule to be detected, which is observed at the spot of each probe on the DNA microarray. There are many times when there is. Therefore, it may not be easy to interpret the test results in samples collected from individual patients with high accuracy only by referring to the test results using typical DNA microarrays for causative bacteria belonging to the same species. Come. In this way, if the test data used for diagnosis is more likely to show a non-negligible shift compared to typical test data measured in the estimated etiology candidate, use the detected data. Therefore, it is desired to propose a method that enables highly accurate diagnosis.

The present invention solves such a problem, and an object of the present invention is to provide a shift in which test data used for diagnosis is not negligible compared to typical test data measured in an estimated etiology candidate. The medical data that can be used to make a high-accuracy diagnosis using the detected data is provided to doctors in charge of diagnosis and treatment of new patients. The object is to provide an electronic medical record system having a function for supporting diagnosis.

  The above-described problem can be solved by constructing an electronic medical chart system having the following configuration.

  The present inventor determines past cases when the test data used for diagnosis may show a non-negligible shift compared to typical test data measured in the estimated etiology candidate. We have scrutinized and found that information on whether or not some of the test data in patients with an estimated etiology showed a shift similar to the test data in new patients was very useful. . That is, if past cases showing similar shifts as test data in a new patient can be selected, the test data in the patient is biased compared to typical test data measured in the estimated etiology candidate. It can be used as a reference case in estimating the cause of the shift. Furthermore, by referring to this similar past case and proceeding with the diagnosis using the detected data, the accuracy of the obtained diagnosis is dramatically improved.

  For the purpose of examining past cases including this examination data, in the electronic medical chart system according to the present invention, patient examination data is stored in a patient electronic medical chart database storing electronic chart information of each patient. Stores clinical findings and treatment data. In addition, new patient examination data, clinical findings data, and past patient examinations recorded in the patient electronic medical record database, which are input as electronic chart information from the input device. Data and clinical findings data are compared, and a mechanism for selecting past cases (patients) that showed similar symptoms and test data is added. In addition, medical record information of selected past cases (patients), that is, examination data, clinical findings data, and treatment data is used as a viewer in the medical field related to diagnosis and treatment of the patient, for example, a doctor in charge of a new patient. In contrast, a mechanism disclosed as a reference case is provided.

That is, the electronic medical record system according to the present invention is
A patient electronic medical record database that stores patient examination data, clinical findings data, and treatment data as electronic medical record information for each patient;
An input device for inputting examination data, clinical findings data, and treatment data as new patient chart information;
Compare the test data and clinical findings data of the new patient input as medical record information with the patient test data and clinical findings data in the past cases stored in the patient electronic medical record database, A similar case patient selection mechanism for selecting one or more patients of past cases having the test data, clinical findings data, similar test data, and clinical findings data of the new patient,
A similar case that is electronically disclosed in a viewable form for doctors involved in the diagnosis and treatment of the new patient by using the similar case patient selection mechanism. An electronic medical record system comprising a patient medical record information disclosure mechanism.

  In the electronic medical record system according to the present invention, patient examination data, clinical findings data, and treatment data are stored in the patient electronic medical record database, and detailed examination data are included in the electronic medical record database. It is possible to select similar past cases (patients) that show high similarity to the test data and clinical findings data of new patients. Furthermore, when diagnosing a new patient, it is more useful by disclosing the examination data, clinical findings data, and treatment data of selected similar past cases (patients) as reference cases to the doctor in charge. It has a diagnosis support function.

The electronic medical record system according to the present invention is:
A patient electronic medical record database that stores patient examination data, clinical findings data, and treatment data as electronic medical record information for each patient;
An input device for inputting examination data, clinical findings data, and treatment data as new patient chart information;
Compare the test data and clinical findings data of the new patient input as medical record information with the patient test data and clinical findings data in the past cases stored in the patient electronic medical record database, A similar case patient selection mechanism for selecting one or more patients of past cases having the test data, clinical findings data, similar test data, and clinical findings data of the new patient,
By the similar case patient selection mechanism, the contents of the chart information of the selected similar case patient can be viewed with respect to the viewer, particularly for doctors involved in the diagnosis and treatment of the new patient The electronic medical record system is characterized by comprising a medical record information disclosure mechanism for patients with similar cases, which is disclosed electronically in FIG.

First, in the electronic medical chart system according to the present invention,
The selection mechanism of the similar case patient is:
It is preferable that one or a plurality of similar case patients to be selected have a ranking function based on similar heights, using as an index the degree of similarity with the test data and clinical findings data of the new patient. In particular, applying the ranking function based on the similar height,
In the similar case patient selection mechanism,
It is desirable to select one patient in the past case having the test data and clinical findings data of the new patient and the most similar test data and clinical findings data.

On the other hand, the medical record information disclosure mechanism for patients with similar cases
Apply non-viewable processing to personal information that is not used for diagnosis and treatment of patients with similar cases and is used only for patient identification, which is included in the medical record information of patients with similar cases disclosed It is desirable to have a function.

The patient electronic medical record database is usually
It is desirable that the detailed contents of the performed examination and the measurement results are included as a part of the examination measurement result database included in the patient examination data constituting the database.

At that time, the detailed contents of the performed test and the measurement result included as part of the test measurement result database included in the patient test data are as follows:
Electronic data indicating the results of probe hybridization reactions for a plurality of DNA probes using a DNA microarray may be included.

For example, as electronic data indicating the result of probe hybridization reaction for a plurality of DNA probes using the DNA microarray,
When the result of the probe hybridization reaction for a plurality of DNA probes to identify the causative bacteria of the infection is indicated by the signal intensity derived from the label,
It may include scan image data of signal intensity derived from a label including spot regions of a plurality of DNA probes, or signal intensity data derived from labels at each spot point of a plurality of DNA probes.

In particular, the details of the tests performed and the measurement results included as part of the database of test measurements included in the patient test data are:
It is preferable to include computerized data indicating the results of probe hybridization reactions for a plurality of DNA probes for identifying the causative bacteria of an infectious disease using a DNA microarray.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a diagram for explaining the concept of a procedure adopted in a mechanism for selecting similar case patients and a chart information disclosure mechanism for similar case patients employed in the electronic medical chart system according to the present invention.

  In the electronic medical record system according to the present invention, patient examination data, clinical findings data, and treatment data are stored on a patient electronic medical record database in accordance with a predetermined formulated format as medical record information of each patient. An index (patient code) indicating that the information is related to each patient is attached to the patient's medical chart information. In addition, since information is added in time series from the start of diagnosis of a patient to the completion of treatment, a time index indicating the recording time of the information is also attached. For example, in various types of examination data, since the date and time when a sample used for a certain examination is collected from a patient is meaningful, the collection date and time of the sample is recorded together with the examination result in addition to the period when the examination result is recorded. Furthermore, in the test data, for the purpose of observing the transition of the patient's symptoms, the result of the test that is repeatedly performed, for example, basic test data such as body temperature, and for the purpose of use for diagnosis, There are specific test data such as blood tests and infectious bacteria tests, for example, as a result of tests performed once. Basic test data is data accumulated in time series, and is recorded separately from specific test data that is a test result that is performed once. In addition, as specific test data, the test results that are carried out on a one-time basis are also recorded corresponding to the raw data obtained from the actual test, and doctors are based on the test results extracted from the raw data. In addition to the judgment results (secondary information) by the person in charge of inspection, if necessary, the raw data recorded as electronic image information and the inspection results (primary information) extracted from the raw data are also viewed. It is possible to do.

  The clinical findings data includes information such as the subjective symptoms of the patient, the course until the symptoms appear, the treatment taken after the symptoms appear, and the procedures obtained by the doctor in charge during the examination. By referring to information such as objective symptoms confirmed by examining the patient, information obtained at the time of examination prior to treatment, and various test results, Diagnosis results, disease names, etc. are recorded. The diagnosis result and the disease name may be corrected to a more appropriate one while proceeding with the treatment. At that time, the correction result is added to the clinical finding data together with the basis for the correction. When all treatments are completed, the etiology that caused the patient's symptoms and the relationship between the etiology and the patient's symptoms were included in the clinical findings data, and past case information and Become. In addition, the patient profile, for example, patient name, age, gender, record of past illness, information on health status other than the medical condition, etc. also constitute a part of clinical finding data.

  The treatment data is determined by the doctor in charge and records the applied treatment information in time series. At the same time, information on changes in the patient's symptoms will be recorded in time series. The treatment given to the patient and the physician's assessment of its effectiveness also form part of the treatment data.

  In the electronic medical record system according to the present invention, patient examination data, clinical findings data, and treatment data are input to the patient electronic medical record database as predetermined formulated information in the patient data input step 101, respectively. Is done. In the form shown in FIG. 1, the patient electronic medical record database stores experimental test data database 102 that stores specific test data that is a test result that is performed once, from the start of diagnosis of a patient to the completion of treatment. In between, clinical data database 103 storing clinical findings data and treatment data to which information is added in time series is divided.

  In the conventional electronic medical record system, the patient electronic medical record database corresponds to the clinical data database 103 that stores the clinical findings data and the treatment data. On the other hand, with regard to test results that are performed on a one-time basis, the judgment results by the tester are attached to each patient's electronic medical record, but when actual raw data needs to be examined, it is separately ordered. ing.

  On the other hand, in the electronic medical record system according to the present invention, the past case recorded in the patient electronic medical record database is recorded in the clinical data database 103 when comparing the new patient's symptoms and test results. In addition to the outline of the test data and clinical findings data, the information by the single test stored in the experimental measurement data database 102 is not only the secondary information but also the primary information stage. Contrast is possible.

  For example, in a test result by probe hybridization reaction for various DNA probes using a DNA microarray, for example, a fluorescent label is attached to a nucleic acid molecule to be detected, and a hybrid is formed at a spot point of each probe. The fluorescence intensity is measured. The measurement results are stored in the experimental measurement data database 102 as primary information electronically recorded, for example, scan image data of a DNA microarray and fluorescence brightness data obtained by extracting the fluorescence brightness of each spot point therefrom. . Next, the determination result obtained by specifying the nucleic acid molecule to be detected based on the fluorescence luminance data of each spot point is recorded as secondary information. Some test data, such as blood sugar concentration and uric acid level, do not need to be further determined from the primary information measurement value and used as secondary information.

  As a method for detecting nucleic acid molecules using a “DNA microarray”, a detection method other than a mode in which a fluorescent label is attached to the nucleic acid molecule to be detected and a probe hybridization method is employed as a detection method. A method is also available. For example, a single-stranded DNA immobilized on a “DNA microarray” is used as a primer, a nucleic acid molecule to be detected is used as a template, and a “single-strand extension reaction” is performed. A form in which a fluorescent label is introduced into the nucleic acid chain extended to the 3 ′ side can also be used. In this form, the specimen sample includes a base sequence complementary to the single-stranded DNA immobilized on the “DNA microarray”, and at the same time forms a base pair with the 3 ′ end of the single-stranded DNA. Only when the nucleic acid molecule to be detected is present, the nucleic acid chain is elongated by the “single-strand elongation reaction”. Using this feature, when there are multiple types of nucleic acid molecules that can form hybrids with single-stranded DNA, select nucleic acid molecules that have a base complementary to the 3'-end base of single-stranded DNA. It can be used as a method of detecting automatically.

In addition, the detection method using a “DNA microarray” exhibits a specific binding ability to a single-stranded DNA comprising a specific base sequence in addition to the detection of a nucleic acid molecule having a base sequence complementary to the single-stranded DNA. A form for detecting proteins and the like can also be used. For example, there is a so-called aptamer as a single-stranded nucleic acid molecule that is not a substrate of the protein itself but exhibits a specific binding ability to the protein. For example, multiple aptamer molecules composed of single-stranded DNA can be immobilized on a “DNA microarray” to detect multiple types of target proteins for which the aptamer molecule exhibits specific binding ability. It is.

Tests that are performed once for the purpose of diagnosis, etc. are performed at the stage when the doctor in charge examines the patient. A valid inspection item is selected. Conventionally, the test result of the selected test item is estimated by judging whether the test result of the patient is valid based on the test result found in a typical case in the estimated etiology candidate. The etiology of the patient is identified from a plurality of etiological candidates.

  Based on the test results found in typical cases, there are some differences from the test results found in typical cases in determining whether the patient's test results are within a reasonable range, In many cases, it is not easy to determine whether or not the range is appropriate. In this case, the past showing the difference from the test results found in typical cases similar to those of new patients, compared with the past cases where the etiology was finally confirmed. It is effective to investigate the presence or absence of these cases (patients) in order to increase the accuracy of diagnosis. That is, even patients with the same etiology often show test results that deviate from test results found in typical cases due to the progress of the symptoms or individual differences.

  In the electronic medical record system according to the present invention, clinical diagnosis data of a new patient, which is input as medical chart information at the stage of diagnosis, is based on the patient's symptom and the symptom input by the doctor in charge. Primary information on the test results, which is the estimated etiology candidate, and which corresponds to the raw data of the test as the test result regarding the test item effective for specifying the etiology candidate, which is input by the department in charge of the test It is. The similar case patient selection mechanism directly compares the test data and clinical findings data of the new patient with the patient test data and clinical findings data of the past cases recorded in the patient electronic medical record database. When comparing clinical findings data of new patients with clinical findings of patients in past cases, the primary information, the symptoms of new patients and the symptoms recorded in patients in past cases are compared. The On the other hand, also in the comparison between the examination data of the new patient and the examination data of the patient in the past case, the primary information of the examination result corresponding to the raw data of the examination is compared.

  In order to achieve the main object of the present invention, first, a plurality of patients in past cases in which test data of new patients and similar test data are recorded are selected. In the process, the primary information of the test results corresponding to the test data of the new patient, particularly the test raw data input by the department in charge of the test; stored in the experimental measurement data 104 and the experimental measurement data database 102 The data matching step 105 compares the experimental measurement data of the past cases (patients). The experimental measurement data of past cases (patients) is sorted in descending order of similarity with the experimental measurement data 104 of new patients, and the experimental measurement data of a group of past cases (patients) having a high sorting order List the index (patient pointer) of the case (patient). Next, in the similar patient clinical data reference step 107, the clinical data database 103 is referred to based on the listed patient pointers 106 of the similar patients, and the clinical findings data of the patients in the past cases (pathology confirmed as symptoms). ) To obtain a working data set. The patient pointer 106 means a series of index information such as a patient code, date and time of recording of examination data, date of experiment (examination), date and time of sampling of an examination sample, etc. in order to specify the chart information of each case (patient).

  A group of past cases (patients) selected as similar case patients are superiors in a list sorted in descending order of similarity, and the number thereof is appropriately determined. For example, when the number of estimated etiology candidate cases is extremely small, a group of past cases (patients) selected as similar case patients are consequently small. On the other hand, when the number of estimated etiological candidate cases is extremely large, as a similar case patient, a group of past cases (patients) selected as a result of a large number of similar cases with the same degree of similarity , Resulting in more. In the medical record information disclosure mechanism of similar case patients, the medical record information of similar case patients selected by this similar case patient selection mechanism, in particular, clinical findings data and treatment data of similar case patients, and diagnosis and treatment of new patients Disclosed electronically in a browsable form for the doctors involved. The disclosure includes information about the etiology that caused the symptoms of patients in similar cases, and the relationship between the etiology and the symptoms that the patient showed, that was finally confirmed when all treatments were completed, and , Information on the relationship between the primary information of the test data and the finally confirmed etiology of a similar case patient is included. This information is often very helpful in diagnosing the etiology of new patients.

  On the other hand, in the medical record information of past cases, information corresponding to personal information of similar case patients is not necessary for comparison of cases, and is removed as non-disclosure information from the working data set. . That is, the process of removing the personal information of the similar patient by the anonymization filter is provided while proceeding from the work data set created in the clinical data reference process 107 to the clinical data display process 109 of the similar patient. . In this process, after the non-disclosure information is sorted in advance by the anonymization filter, only the information that has passed through the filter is disclosed with a doctor who is involved in the diagnosis and treatment of a new patient as a browsing target person. At the same time, treatment data for patients with similar cases is also disclosed, which is often useful as a reference in determining a treatment policy to be applied to a new patient after diagnosis.

A similar case patient selection mechanism and a similar case patient medical record information disclosure mechanism included in the electronic medical record system according to the present invention refer to, for example, examination data obtained by state-of-the-art medical examination such as genetic examination. It is effective in advancing diagnosis. For example, when a test is performed to identify the causative bacteria of an infectious disease using a DNA microarray, even if the causative bacteria of the infectious disease are actually the same, depending on the situation, each probe of the DNA microarray to be measured The fluorescence brightness data may be different. In many cases, a simple judgment cannot be made simply based on the fluorescence luminance data of each probe of the DNA microarray to be measured. If, in the past cases, the fluorescence intensity data of each probe of a DNA microarray that is very similar were measured for the causative bacteria of the finally confirmed infection, the test data was compared with this result. The accuracy of the determination can be dramatically increased.

FIG. 2 shows a basic configuration of an information processing apparatus that can be used for information processing operations in a similar case patient selection mechanism and a similar case patient medical record information disclosure mechanism included in the electronic medical chart system according to the present invention. FIG.

  The electronic medical record system is constructed by using an information processing apparatus including an external storage device 201, a central processing unit (CPU) 202, a memory 203, and an input / output device 204. The external storage device 201 is used to store programs and the like used for various information processing processes in the patient electronic medical record database, the similar case patient selection mechanism, and the similar case patient medical record information disclosure mechanism. The central processing unit (CPU) 202 is used for information processing and information management processes executed in the system. The memory 203 can be used for temporarily recording programs, subroutines, and data when executing information processing and information management processes via a central processing unit (CPU) 202. The input / output device 204, when executing the process of inputting data recorded in the patient electronic medical record database, information processing, and information management, for example, an input operation such as parameter control set externally when executing a program, Various display and output operations are performed. For example, when operating a similar case patient selection mechanism and a similar case patient chart information disclosure mechanism, the user (doctor) is interacted via the input / output device 204.

The electronic medical record system is a form in which a large number of users (doctors) use the system in parallel, and at the same time, the patient electronic medical record database stored in the external storage device is unified on the entire system. The shape is managed. Therefore, in many cases, a client-server type system configuration shown in FIG. 3 is used. The patient electronic medical record database is stored on a server shared by many clients. The patient electronic medical record database itself is stored in a server after being divided into an experimental measurement data database 102 and a clinical data database 103. A large number of clients are independently used by, for example, an examination department in charge of inputting and managing experimental measurement data in the experimental measurement data database 102 and a group of doctors in charge of patient diagnosis and treatment. In addition, when operating the similar case patient selection mechanism and the similar case patient chart information disclosure mechanism, the use target is limited to doctors, but each doctor interacts with the system through each client.

(Preferred embodiment)
In the following, the selection mechanism of similar case patients provided in the electronic medical record system according to the present invention, the medical record information disclosure mechanism of similar case patients, in particular, the inspection data of new patients in the selection mechanism of similar case patients, and similar A process of selecting a plurality of patients in the past cases in which examination data to be recorded is recorded will be described with a specific example. At that time, in the data matching step shown in FIG. 1, the experimental measurement data are compared with each other. As an example of the experimental measurement data, a nucleic acid molecule to be detected contained in a test sample using a DNA microarray. The inspection data that has been detected will be described as an example.

In addition to genetic testing using a DNA microarray, for example, genetic testing using genomic analysis techniques such as quantitative PCR can be used to compare experimentally measured data with each other in the same manner. Furthermore, with regard to the state-of-the-art medical examination technology, which is very difficult to interpret experimental measurement data, the experimental measurement data is not interpreted, and the experimental measurement data is compared with each other. Therefore, the effect of the present invention is exhibited.

That is, the example described below is an example of the best embodiment according to the present invention, but the present invention is not limited to such an embodiment.

FIG. 4 shows the detection of nucleic acid molecules contained in a “sample” 401 by applying a probe hybridization method using a DNA microarray in which multiple types of DNA probes are immobilized in an array. It is a figure which shows typically the flow of the process to do. “Sample” 401 is a liquid sample or a solid sample presumed to contain a nucleic acid molecule to be detected. For example, when it is presumed that the patient suffers from an infectious disease, the sample is a sample containing the presumed causative agent of the infectious disease. In fact, it includes possible body fluids such as blood, sputum, gastric juice, vaginal secretions, mucus in the mouth, and excreta such as urine and feces that can be collected from patients A sample 401 is selected as the one expected to be recorded.

  Next, a nucleic acid molecule derived from a causative bacterium that is used to identify a causative bacterium of an infectious disease presumed to be contained in the sample 401 is, as necessary, a “biochemical amplification” step 402. Amplify. For example, there is a difference in the base sequence of 16s rRNA depending on the type of each bacterium, and it is possible to identify a bacterium by using this base sequence difference. When 16s rRNA having a unique base sequence is to be detected for each type of bacteria, genomic DNA is extracted from the bacteria and PCR amplification is performed only on the region encoding the 16s rRNA. Is available. In most of the causative bacteria of infectious diseases, the base sequence of the region encoding 16s rRNA has been elucidated, and primers for PCR amplification of the region encoding 16s rRNA are designed based on the known base sequence. A PCR amplification product corresponding to a region encoding 16s rRNA is prepared using genomic DNA extracted from bacteria as a template and the PCR amplification primer. In addition to the PCR amplification method, other amplification methods such as the LAMP method can be applied as a method for amplifying nucleic acid molecules.

  When detecting a nucleic acid molecule to be detected by applying the probe hybridization method, it is desirable that the concentration of the nucleic acid molecule to be detected contained in the hybridization reaction solution is a certain level or higher. Therefore, if necessary, a sample DNA solution having a nucleic acid molecule concentration to be detected of a certain level or more can be prepared by further performing PCR amplification using the DNA fragment of the primary amplification product as a template.

  On the other hand, the detection of the hybrid formation between the DNA probe and the nucleic acid molecule to be detected is performed by labeling the nucleic acid molecule to be detected itself, and the nucleic acid molecule to be detected immobilized on the DNA microarray along with the formation of the hybrid. This is done via the signs attached to Therefore, the nucleic acid molecule to be detected contained in the sample 401 or the nucleic acid molecule of the amplification product amplified in the biochemical amplification step 402 is used in the “label mixing” step 403 using various labeling techniques. Give a sign. As a labeling substance used for this labeling, it is preferable to use a fluorescent labeling substance that is widely used, for example, a fluorescent substance such as Cy3, Cy5, or Rhodamin. Alternatively, an intercalator-type fluorescent labeling substance can be attached to the nucleic acid molecule of the amplification product via a linker. When these fluorescent labels are used, the amount of nucleic acid molecules to be detected to be immobilized in accordance with the formation of a hybrid from the spot points of the DNA probes immobilized on the DNA microarray, as a whole DNA microarray, depending on the fluorescence intensity, It can be observed as a two-dimensional image. In addition, labeling using various labeling methods can be performed in the “label mixing” step 403, or can be introduced when the nucleic acid chain is extended during the biochemical amplification step 402.

  Preliminarily design the probe sequence, immobilize the DNA probe produced by chemical synthesis, and prepare it in the step 404 of constructing the DNA microarray. The DNA probe immobilized using the DNA microarray chip and “label mixing” The target nucleic acid molecule labeled in step 403 is reacted in a hybridization reaction step 405.

  In step 404 of forming a DNA microarray, a plurality of types of DNA probes are spotted and immobilized in an array on the surface of the probe fixing carrier according to a predetermined arrangement order. For the surface of the probe-immobilizing support itself, a material that does not adsorb target nucleic acid molecules non-selectively is selected. For example, it is preferable to use a flat substrate such as a glass substrate, a plastic substrate, or a silicon wafer as a probe fixing carrier (substrate). If it is possible to spot and fix multiple types of DNA probes in an array according to a predetermined arrangement order, in addition to a flat substrate, a three-dimensional structure with irregularities, a spherical shape such as a bead , Rods, strings, threads, etc. may be used.

  When a material that does not adsorb target nucleic acid molecules in a non-selective manner is selected on the surface of the probe immobilization carrier (substrate) itself, the DNA probe itself is densely adsorbed on the surface of the immobilization carrier (substrate). It is difficult to fix. Therefore, the DNA probe is usually immobilized on the surface of the immobilizing carrier (substrate) by selecting a form in which the two are linked via a covalent bond. Specifically, the surface of the carrier for immobilization (substrate) is subjected to a surface treatment in which a functional group that can be used for immobilizing a DNA probe is introduced in advance, while the functional group having reactivity is also present on the DNA probe side. Is preferably introduced in advance, and a form of linking via a covalent bond is selected by a reaction between the two. As a result of the DNA probe being stably immobilized on the surface of the carrier (substrate) for immobilization, when the DNA probe and the target nucleic acid molecule form a hybrid, the labeled target nucleic acid molecule is spotted on the DNA probe. Fixed to a point quantitatively. In this state, since the amount of the labeled target nucleic acid molecule is detected by using the label, the quantitativeness of the detection becomes high and a preferable form is obtained.

  An example of means for stably immobilizing a DNA probe on the surface of this immobilizing carrier (substrate) is as follows. Maleimide groups are introduced into the surface of the immobilizing carrier (substrate), while the DNA probe ends. After the introduction of a thiol (-SH) group, the reaction is carried out by the maleimide group and the thiol (-SH) group, whereby the immobilization is achieved by a covalent bond. For example, an aminosilane coupling agent is reacted with the surface of the glass substrate to form a fixing layer of the coupling agent. Next, by reacting an amino group derived from the coupling agent with an EMCS reagent (N- (6-Maleimidocaproyloxy) succinimide: Dojin) and causing a reaction between the succinimide moiety and the amino group, maleimide derived from the EMCS reagent A group is introduced. The SH group is introduced into the single-stranded DNA by 5′-Thiol-Modifier C6 (on the 5 ′ end of the DNA strand having the desired base sequence on a DNA automatic synthesizer when the single-stranded DNA is chemically synthesized. By using (Glen Research), a thiol (-SH) group is introduced into the terminal via a linker.

  In addition, an epoxy group is introduced on the surface of the fixing carrier (substrate), while an amino group is introduced at the 3 ′ end of the single-stranded DNA, and a bond is formed by the reaction of this amino group and the epoxy group. Is available. In addition, surface treatment with various silane coupling agents can be suitably used for introduction of the functional group onto the surface of the fixing carrier (substrate). A method of using a functional group introduced by the silane coupling agent and a functional group introduced at the end of the single-stranded DNA and linking them can be used. Furthermore, it is also possible to use a form in which a functional group is introduced onto the surface of a fixing carrier (substrate) using a method of coating a resin having a functional group.

  On the other hand, a base sequence complementary to a part of the base sequence of the nucleic acid molecule to be detected is used as the base sequence of the DNA probe. For example, for the purpose of identifying the causative bacterium of an infectious disease, a DNA fragment of an amplification product having a base sequence corresponding to the genomic portion coding for 16s rRNA of the bacterium is used as a nucleic acid molecule to be detected. That is, a DNA probe corresponding to the partial base sequence is designed from the base sequence of the coding region of 16s rRNA. The partial base sequence used for the DNA probe is preferably selected to have a characteristic characteristic of the bacterium and a very high specificity. In addition, it is necessary to confirm that the nucleic acid molecule to be detected retains the entire base sequence of the 16s rRNA coding region of the bacterium, and a plurality of nucleic acid molecules included in the base sequence of the 16s rRNA coding region of the bacterium A plurality of types of DNA probes having partial base sequences corresponding to the positions are designed. At that time, the base length and base sequence of each DNA probe can be selected so that the probability of forming a hybrid with a nucleic acid molecule to be detected does not vary as much as possible. preferable.

  For example, when there are a plurality of candidate causative bacteria of an infectious disease that a patient may have, for example, each of the causative fungus candidates, for example, the 16s rRNA coding region of the fungus A plurality of types of DNA probes having partial base sequences corresponding to a plurality of locations included in the base sequence are designed. On the DNA microarray, a plurality of types of DNA probes designed for identification of causative bacteria of each infectious disease are immobilized in an array, and as a whole, causative bacteria of each infectious disease A probe array for identifying candidates is arranged in a two-dimensional matrix regularly arranged for the types of candidates of causative bacteria causing infection.

In the hybridization reaction step 405, after the hybridization reaction is completed, the surface of the DNA microarray is washed to remove the liquid containing nucleic acid molecules to be detected. In addition, nucleic acid molecules non-selectively attached to the surface of the DNA microarray are washed away. When a fluorescent label is used as the label, the washed DNA microarray is usually drained and dried, and the process proceeds to the next fluorescence measurement step 406. In the fluorescence measurement step 406, the amount of nucleic acid molecules to which the fluorescent label that has been immobilized is applied in association with the formation of the hybrid with respect to the spot points of the DNA probes immobilized on the DNA microarray, and the amount of fluorescence from the fluorescent labeling substance is determined. Measure and quantify. At that time, the spot points of the DNA probes fixed on the DNA microarray are arranged in an array. The entire surface of the DNA microarray is irradiated with excitation light, and the fluorescence intensity of the spot points arranged in the array is measured. Record as a two-dimensional image. That is, in the scan image process 407, the fluorescence light measurement sensor is relatively two-dimensionally scanned while irradiating the entire surface of the DNA microarray with excitation light, and is recorded as two-dimensional image information.

Next, the principle of a method for identifying the causative bacteria of an infectious disease using a DNA probe immobilized on a DNA microarray will be described. FIG. 5 schematically shows, as an example, a procedure for distinguishing S. aureus from other bacteria using a DNA microarray prepared for the purpose of identifying S. aureus and its principle.

  In FIG. 5, the left column is a detection processing sequence of a nucleic acid molecule containing the base sequence of the coding region of 16s rRNA derived from S. aureus wild strain, and the right column is the coding region of 16s rRNA derived from E. coli wild strain. It is the detection processing series of the nucleic acid molecule containing the base sequence. For example, the left corresponds to a flow of processing a blood sample collected from a patient infected with S. aureus, and the right corresponds to a flow of processing a blood sample collected from a patient infected with E. coli.

  In both processes, the same process is basically performed in each step of extracting contained DNA, amplifying a nucleic acid molecule to be detected, and applying a fluorescent label from a collected sample. That is, first, DNA is extracted from a collected sample, for example, blood or sputum of a bacterially infected patient. In this step, all genomic DNA contained in the collected sample is extracted. In addition, in the collected sample, in addition to the causative bacteria of the infectious disease, the patient's own cells, for example, various blood cells and somatic cells may be mixed, and generally extracted. DNA may also include human DNA derived from patient somatic cells.

  When the content of the nucleic acid molecule to be detected derived from the causative agent of the infectious disease is low in the total amount of DNA extracted from the sample, the nucleic acid molecule to be detected is selectively amplified using a PCR method or the like. Amplify. In this amplification step, a labeling step is generally performed in which a fluorescent substance or a substance capable of binding the fluorescent substance is added as a label to the amplification product.

  When amplification is not performed, a fluorescent substance or a substance capable of binding the fluorescent substance is directly added to the extracted DNA as a label. Alternatively, the extracted DNA can be used as a template to prepare a complementary strand, and in the process, a fluorescent substance or a substance capable of binding the fluorescent substance can be added as a label to the extended DNA chain. Good.

  In order to identify the causative bacterium of the infectious disease, a DNA portion having a base sequence specific to the bacterium is selected as the nucleic acid molecule to be detected. For example, a so-called 16s rRNA base sequence of ribosome RNA used for classification of bacteria contains a characteristic base sequence that enables discrimination of each bacterium. Therefore, when performing amplification, it is common to prepare a DNA fragment containing the base sequence of the coding region of 16s rRNA as a PCR amplification product from the genomic DNA of the bacterium.

  Usually, the type of infectious disease-causing bacteria contained in the collected sample is unknown, and a DNA fragment containing the base sequence of the coding region of human-derived 16s rRNA is not amplified. Regarding causative bacteria, PCR primers that can simultaneously amplify a DNA fragment containing the base sequence of the coding region of 16s rRNA derived from many types of bacteria are used. In other words, among various causative bacteria of infectious diseases, by using a multiplex PCR reaction using a similar partial primer sequence in the genomic DNA and corresponding mixed primer sets, Conditions are selected under which amplification of a DNA fragment containing the base sequence of the 16s rRNA coding region derived from the species is simultaneously performed.

  In addition, when it is desired to perform a more detailed analysis of the base sequence of the specified causative agent of the infectious disease, for example, a PCR primer set dedicated to S. aureus and a PCR primer set dedicated to E. coli are separately selected. By using the PCR primer set dedicated to each bacterium, only a specific portion in the genomic DNA of the target bacterium can be selectively amplified. On the other hand, in the sample DNA solution for hybridization reaction containing the PCR amplification product obtained by the multiplex PCR reaction, the types of base sequences of the contained DNA fragments are very limited.

  In the collected sample, the abundance ratio of the causative bacteria of the infectious disease is significantly high, but it is a small amount, but it is not rare that bacteria existing in nature are mixed. As a result of the multiplex PCR reaction, in the sample DNA solution for the hybridization reaction, in addition to the DNA fragment derived from the causative bacterium of the infectious disease to be detected, there is also a slight amount of DNA fragment derived from the contaminated natural bacteria. Often included. In addition, the causative bacteria of the infectious disease to be detected is not a single strain that exists in nature, and there are often several types of clinically isolated strains. In the collected sample, a plurality of strains may coexist with respect to the causative agent of the infectious disease to be detected. By performing PCR amplification, the types of base sequences of DNA fragments contained in the sample DNA solution for hybridization reaction are very limited, but rarely only one type.

  The DNA probe designed for the purpose of determining S. aureus is a hybrid with a single-stranded DNA containing the base sequence of the 16s rRNA coding region derived from S. aureus. In the system, a nucleic acid molecule containing a label is observed (positive) at a spot point of a DNA probe for S. aureus determination on a DNA microarray. On the other hand, when a DNA probe designed for the purpose of determining S. aureus has extremely high selectivity, it is a hybrid with a single-stranded DNA containing the base sequence of the coding region of 16s rRNA derived from the wild-type E. coli strain. In the processing system on the right side of FIG. 5, no nucleic acid molecule containing a label is observed (negative) at the spot of the DNA probe for S. aureus determination on the DNA microarray.

  In addition, since the DNA probe designed for the purpose of determining E. coli constitutes a hybrid with a single-stranded DNA containing the base sequence of the 16s rRNA coding region derived from E. coli, the right processing system in FIG. On the DNA microarray, a nucleic acid molecule containing a label is observed (positive) at the spot point of the DNA probe for E. coli determination. On the other hand, when a DNA probe designed for the purpose of determining Escherichia coli has extremely high selectivity, it is a hybrid with a single-stranded DNA containing the base sequence of the coding region of 16s rRNA derived from S. aureus wild strains. In the processing system on the right side of FIG. 5, no nucleic acid molecule containing a label is observed (negative) at the spot point of the DNA probe for E. coli determination on the DNA microarray.

When DNA probes designed for the purpose of determining the causative bacteria of various infectious diseases have extremely high selectivity, a plurality of DNA probes for determining the causative bacteria of these various infectious diseases are placed on the DNA microarray. It can be spotted in an array according to a predetermined arrangement order, and can be used to determine the presence / absence of the causative bacteria of the infectious disease at once.

When applying the method and principle of identifying the type of causative bacteria of the infectious disease contained in the sample described in FIG. 4 and FIG. 5 above, the actual operation, its conditions, etc. will be described in more detail below. An example will be described.

<Preparation of probe DNA>
As probes for detecting Enterobacter cloacae, probes I-1 to I-7 having the nucleic acid sequences (In) (n is a positive integer) shown in Table 1 were designed.

  Specifically, the base sequences of probes I-1 to I-7 shown in Table 1 below are selected from the genome portion coding the 16s rRNA of the bacterium. The base sequences of these probe groups are very specific to the bacteria, and at the same time, each probe was hybridized with genomic DNA as a template and cDNA prepared from the 16s rRNA coding region. It is designed by selecting the base length and the content ratio of each base so that it can be expected that the hybridization efficiency does not vary as much as possible.

  In each DNA probe, after chemical synthesis, a thiol group is introduced into the 5 ′ end of the nucleic acid strand as a functional group used for immobilization on the solid phase surface in a DNA microarray according to a conventional method. After introduction of the functional group at the 5 'end, each DNA probe is purified and lyophilized. The freeze-dried DNA probe for detecting Enterobacter cloacae is stored in a freezer at −30 ° C.

  Further, in order to detect various types of pathogenic bacteria, a probe having a very high specificity for the bacteria is designed from the genome portion encoding 16s rRNA derived from each bacteria using the same technique. In Tables 2 to 10, DNA probes for detecting S. aureus: An, DNA probes for detecting Staphylococcus epidermidis: Bn, DNA probes for detecting E. coli: Cn, DNA probes for detecting Klebsiella pneumoniae: D- n, DNA probe for detecting Pseudomonas aeruginosa: En, DNA probe for detecting Serratia bacteria: Fn, DNA probe for detecting Streptococcus pneumoniae: Gn, DNA probe for detecting Haemophilus influenzae: Hn, and Enterococcus A DNA probe for detecting Fecalis: Jn (where n is a positive integer) represents a selected base sequence, respectively.

<Preparation of PCR primers for sample DNA amplification>
In order to detect 10 species of the above-mentioned pathogenic bacteria, a region encoding 16s rRNA (target nucleic acid) derived from the bacteria is amplified using genomic DNA as a template. At that time, primers having the nucleic acid sequences shown in Table 11 were designed as PCR primers for amplification that can be shared by various pathogenic bacteria.

  Specifically, in order to selectively amplify only the coding region of 16s rRNA having a length of about 1500 bases on the genomic DNA, partial bases showing high commonality at both ends of the coding regions of the above-mentioned 10 types of pathogenic bacteria Based on the sequence, when forming a hybrid with the template DNA, a base sequence having the same melting temperature as possible is selected. In addition, 3 types of primers each containing a mutation were designed so that mutant strains and a plurality of 16s rRNA coding regions existing on the genome could be amplified simultaneously.

Each primer shown in Table 11 is purified by high performance liquid chromatography (HPLC) after synthesis. As the primer solution, three kinds of Forward Primer and three kinds of Reverse Primer were mixed, and each Primer concentration was dissolved in TE buffer so that the final concentration would be 10 pmol / μl.

<Extraction of fungal genome>
(Pre-processing for Genome extraction)
1.0 ml (OD ₆₀₀ = 0.7) of a solution containing microorganisms is collected in a 1.5 ml capacity microtube and centrifuged (8500 rpm, 5 min, 4 ° C.) to collect the cells. After discarding the supernatant, 300 μl of Enzyme Buffer (50 mM Tris-HCl: pH 8.0, 25 mM EDTA) is added, and the cells are resuspended using a mixer. The resuspended bacterial solution is centrifuged again (8500 rpm, 5 min, 4 ° C.) to collect the bacterial cells. After discarding the upper sperm, the following dissolved enzyme solution is added to the collected cells and resuspended using a mixer.

Lysozyme 50 μl (20 mg / ml in Enzyme Buffer)
N-Acetylmuramidase SG 50 μl (0.2 mg / ml in Enzyme Buffer)

Next, a lytic enzyme solution is added, and the resuspended bacterial solution is allowed to stand for 30 minutes in a 37 ° C. incubator to lyse the cell wall.

(Genome extraction)
After the lysis treatment, Genome DNA extraction of each microorganism is performed by the following procedure using a commercially available nucleic acid purification kit (MagExtractor-Genome-: manufactured by TOYOBO).

[Step 1]
First, after the dissolution treatment, 750 μl of the dissolution / adsorption solution and 40 μl of the magnetic bead solution are added to the microorganism suspension, and vigorously stirred for 10 minutes using a tube mixer. By this operation, genomic DNA is adsorbed on the surface of the magnetic bead particles.

[Step 2]
Next, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect the magnetic bead particles on the wall surface of the tube. After that, throw away the fine spirit while it is still on the stand.

[Step 3]
Next, 900 μl of the washing solution is added, and the mixture is stirred for about 5 seconds with a mixer to resuspend the magnetic bead particles.

[Step 4]
Again, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect the magnetic bead particles on the wall of the tube. After that, throw away the fine spirit while it is still on the stand.

[Step 5]
Steps 3 and 4 are repeated to perform the second cleaning.

[Step 6]
Add 900 μl of 70% ethanol and stir with a mixer for about 5 seconds to resuspend the magnetic bead particles.

[Step 7]
Next, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect the magnetic bead particles on the wall surface of the tube. After that, throw away the fine spirit while it is still on the stand.

[Step 8]
Steps 6 and 7 are repeated to perform a second 70% ethanol wash.

[Step 9]
Add 100 μl of pure water to the collected magnetic bead particles, and stir with a tube mixer for 10 minutes. By this operation, the genomic DNA adsorbed on the magnetic bead particle surface is eluted.

[Step 10]
Next, the microtube is set on a separation stand (Magnetic Trapper) and allowed to stand for 30 seconds to collect the magnetic bead particles on the wall surface of the tube. Thereafter, the supernatant containing the dissolved genomic DNA is collected in a new tube while being set on the stand.

(Quality test of recovered Genome DNA)
Genome DNA of the collected microorganisms is subjected to agarose electrophoresis and absorbance measurement at 260 nm / 280 nm according to a conventional method, and the quality (contamination amount of low-molecular nucleic acid, degree of degradation) is assayed, and the amount of recovered DNA (containing) (Concentration) is calculated.

  The recovered Genome DNA is dissolved in TE buffer so as to have a final concentration of 50 ng / μl, and used as a template DNA solution in the PCR amplification reaction described below.

In this example, degradation of Genome DNA and contamination with rRNA were not observed.

<Production of DNA microarray>
[1] Cleaning of glass substrate Place a synthetic quartz glass substrate (size: 25 mm (W) x 75 mm (L) x 1 mm (T), manufactured by Iiyama Special Glass Co., Ltd.) in a heat-resistant and alkali-resistant rack to a predetermined concentration. Immerse in the prepared cleaning solution for ultrasonic cleaning. After soaking in the cleaning solution overnight, ultrasonic cleaning is performed for 20 minutes. Subsequently, the substrate is taken out, rinsed lightly with pure water, and then subjected to ultrasonic cleaning in ultrapure water for 20 minutes. Next, the substrate is immersed in a 1N sodium hydroxide aqueous solution heated to 80 ° C. for 10 minutes. Again, pure water cleaning and ultrasonic cleaning in ultra pure water are performed to obtain a cleaned quartz glass substrate for DNA microarray chip fabrication.

[2] Surface treatment A silane coupling agent KBM-603 (manufactured by Shin-Etsu Silicone) is dissolved in pure water to a concentration of 1% and stirred at room temperature for 2 hours. Subsequently, the cleaned quartz glass substrate is immersed in an aqueous silane coupling agent solution and left at room temperature for 20 minutes. The quartz glass substrate is pulled up, and the surface is lightly rinsed with pure water, and then blown dry by blowing nitrogen gas on both sides of the substrate. Next, the substrate blown with nitrogen is baked in an oven heated to 120 ° C. for 1 hour to complete the coupling agent treatment. By this coupling agent treatment, amino groups derived from the aminosilane coupling agent are introduced into the surface of the quartz glass substrate.

Subsequently, N-maleimidocaproyloxy succinimide (N- (6-Maleimidocaproxy) succinimido; hereinafter abbreviated as EMCS) manufactured by Dojindo Laboratories Ltd. was added to the final concentration in a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol. Prepare an EMCS solution that is dissolved to a concentration of 0.3 mg / ml. The quartz glass substrate after baking is allowed to cool and immersed in the prepared EMCS solution at room temperature for 2 hours. By this treatment, the amino group introduced to the surface by the silane coupling agent treatment and the succinimide group of EMCS react to introduce a maleimide group on the surface of the quartz glass substrate. The quartz glass substrate pulled up from the EMCS solution is washed with a 1: 1 mixed solvent of dimethyl sulfoxide and ethanol, further washed with ethanol, and then dried in a nitrogen gas atmosphere.

[3] Probe DNA
Dissolve each of the probe for detection of pathogenic bacteria prepared above in pure water, dispense each to a final concentration (at the time of ink dissolution) of 10 μM, and then freeze-dry to remove moisture.

[4] Probe DNA ejection by a BJ printer and binding to a substrate 7.5% by weight of glycerol, 7.5% by weight of thiodiglycol, 7.5% by weight of urea, 1.0% by weight of acetylenol EH (manufactured by Kawaken Fine Chemical Co., Ltd.) Prepare an aqueous solution. Subsequently, the seven types of probes I-1 to I-7 (Table 1) prepared above are dissolved in the aqueous solution so as to have a specified concentration. The obtained DNA solution is filled in an ink tank for a bubble jet printer (trade name: BJF-850 manufactured by Canon Inc.) and mounted on a print head.

  The bubble jet printer used here is modified so that printing on a flat plate is possible. In addition, this bubble jet printer has an apparatus configuration capable of spotting a droplet of about 5 picoliters of DNA solution at a pitch of about 120 micrometers by inputting a print pattern according to a predetermined file creation method. It has become.

Subsequently, using this modified bubble jet printer, a printing operation is performed on the surface of one quartz glass substrate to produce a DNA microarray. After confirming that each DNA solution was spotted reliably, it was left in a humidified chamber for 30 minutes to react the maleimide group on the quartz glass substrate surface with the thiol group at the 5 ′ end of the DNA probe. Let

[5] Washing After the reaction for 30 minutes, the solution containing unreacted DNA remaining on the surface is washed away with 10 mM phosphate buffer (pH 7.0) containing 100 mM NaCl. As a result, a DNA microarray chip is obtained in which single-stranded DNAs for probes are immobilized in an array on the surface of a quartz glass substrate.

<Amplification and labeling of sample DNA (PCR amplification & incorporation of fluorescent label)>
Amplification of the region encoding 16s rRNA (target nucleic acid) derived from the microorganism using the genomic DNA extracted from the microorganism collected from the sample as a template, and labeling of the amplification product using a fluorescent label in the following Perform under the PCR reaction conditions indicated.

  The temperature cycle is performed using a commercially available thermal cycler.

After the PCR amplification reaction, the primer is removed using a commercially available purification column (QIAGEN QIAquick PCR Purification Kit), and then the amplification product is quantified. The obtained amplification product is fluorescently labeled derived from Cy-3 dUTP, and is used as labeled sample DNA in the following hybridization reaction.

<Hybridization>
A probe using a DNA microarray chip prepared according to the procedure of <Preparation of DNA microarray>, and labeled sample DNA prepared according to the procedure of <Amplification and labeling of sample DNA (PCR amplification & incorporation of fluorescent label)> -The sample DNA is detected by a hybridization reaction.

(Blocking of DNA microarray)
BSA (bovine serum albumin Fraction V: manufactured by Sigma) is dissolved in 100 mM NaCl / 10 mM Phosphate Buffer so as to be 1 wt%. In this BSA solution, the DNA microarray chip prepared according to the procedure of <Preparation of DNA microarray> is immersed at room temperature for 2 hours to perform blocking treatment. After completing the blocking treatment, a 2 × SSC solution containing 0.1 wt% SDS (sodium dodecyl sulfate) (NaCl 300 mM, sodium citrate dihydrate, C ₆ H ₅ Na ₃ .2H ₂ O) 30 mM, pH 7.0 ). Further, after rinsing with pure water, draining is performed with a spin dryer.

(Hybridization)
The drained DNA microarray chip is set in a hybridization apparatus (Genomic Solutions Inc. Hybridization Station), and a hybridization reaction is carried out under the following conditions.

[Hybridization solution]
6 × SSPE / 10% Formamide / Target (2nd PCR Products total amount)
(6 × SSPE: NaCl 900 mM, NaH ₂ PO ₄ .H ₂ O 60 mM, EDTA 6 mM, pH 7.4)
[Hybridization conditions]
65 ° C. 3 min → 92 ° C. 2 min → 45 ° C. 3 hr → Wash 2 × SSC / 0.1% SDS at 25 ° C. → Wash 2 × SSC at 20 ° C. → (Rinse with H ₂ O: Manual) → Spin dry

<Detection of microorganism-derived target DNA (fluorescence measurement)>
After completion of the hybridization reaction, the fluorescence intensity at each spot point on the drained DNA microarray chip is measured using a fluorescence detection apparatus for DNA microarray (Axon, GenePix 4000B). At that time, the fluorescence intensity at each spot point on the DNA microarray is recorded as two-dimensional image information.

FIG. 6 shows an example of a two-dimensional image representing the fluorescence intensity at each spot point on the DNA microarray. In FIG. 6, the fluorescence intensity resulting from the fluorescently labeled DNA that hybridizes to the DNA probe fixed to each spot point is displayed as a darker color as the intensity increases. That is, as the amount of fluorescently labeled DNA to be hybridized with respect to the DNA probe fixed to each spot point is larger, it is displayed as a darker color.

  On the DNA microarray illustrated in FIG. 6, DNA probes for detection of 10 types of microorganisms described in <Preparation of probe DNA>; Staphylococcus aureus: An, Staphylococcus epidermidis: Bn, E. coli: C -N, Klebsiella pneumoniae: Dn, Pseudomonas aeruginosa: En, Serratia bacteria: Fn, Streptococcus pneumoniae: Gn, Haemophilus influenzae: Hn, Enterobacter cloacae: In, And Enterococcus faecalis: Jn are each spotted in an array.

A sample sample containing an amplification product of a region encoding 16s rRNA (target nucleic acid) derived from the bacteria is reacted with the probe DNA immobilized on the DNA microarray using genomic DNA extracted from various pathogenic bacteria as a template. . In FIG. 6, a scan image 601 of the DNA microarray on the left is an example of a two-dimensional image observed when a specimen sample containing an amplification product of a region encoding 16s rRNA (target nucleic acid) derived from Staphylococcus aureus is reacted. The right DNA microarray scan image 602 is an example of a two-dimensional image observed when a specimen sample containing an amplification product of a region encoding 16s rRNA (target nucleic acid) derived from E. coli is reacted. is there.

In the scan image 601 of the DNA microarray, the fluorescence intensity caused by the fluorescent label at each spot point of the DNA probe for detecting Staphylococcus aureus: An increases, while in the scan image 602 of the DNA microarray, the DNA for detecting E. coli is used. The fluorescence intensity resulting from the fluorescent label at each spot point of the probe: Cn is high. Ideally, each of the plurality of DNA probes for detecting the causative fungus forms a hybrid only with the amplification product of the region encoding 16s rRNA (target nucleic acid) derived from the bacteria, and 16s rRNA derived from other bacteria The base sequence of the DNA probe is selected so that it does not form a hybrid with the amplification product of the region encoding (target nucleic acid).

  However, actually, it is necessary to select a plurality of types of DNA probes for detecting each causative bacterium so as to cover the entire amplification product of the region encoding 16s rRNA (target nucleic acid) derived from the bacterium, Some DNA probes have base sequences capable of misfit hybridization with amplification products of regions encoding 16s rRNA (target nucleic acid) derived from other bacteria. That is, as a result of the so-called “cross-hybridization reaction”, the amplification product of the region encoding 16s rRNA (target nucleic acid) derived from the target pathogenic fungus is hybridized with some other DNA probes for detecting bacteria. The phenomenon that forms the body occurs. In the scan image 601, there are several spots where the fluorescence intensity due to the fluorescent label is observed, in addition to the spot points of the S. aureus detection DNA probe: An. Similarly, in the scan image 602, there are several spots where the fluorescence intensity due to the fluorescent label is observed in addition to the spot points of the Escherichia coli detection DNA probe: Cn.

  On the other hand, when the fluorescence intensity at each spot point of the DNA probe for detection of E. coli: Cn in the scan image 602 is compared in detail, some of them have inferior fluorescence intensity compared to other spots. ing. As a means for attaching a fluorescent label to an amplification product of a region encoding 16s rRNA (target nucleic acid) derived from the target Streptococcus, a method of linking Cy-3 dU instead of T when extending a DNA strand Since it is used, the hybridization efficiency between the portion containing Cy-3 dU and the DNA probe is significantly lower than when it originally contains T. In addition, in the amplification product of the region encoding 16s rRNA (target nucleic acid) derived from the target Streptococcus, there is a product that does not contain a fluorescent label without Cy-3 dU linked in place of T. Yes. For this reason, depending on the base sequence of the DNA probe, there are few hybrid formations with DNA fragments that are actually labeled with a fluorescent label, whereas hybridization with DNA fragments without a fluorescent label is relatively superior. May occupy. In that case, there is no significant difference in the total amount of hybrids formed at the spot point of the DNA probe, but the fluorescence intensity due to the fluorescent label observed at the spot point is inferior.

  In addition, for individual pathogenic bacteria, although the clinically isolated strain belongs to the same species, a typical wild-type strain based on the base sequence in designing the DNA probe for detecting the pathogenic bacteria is used. In many cases, the strain corresponds to a mutant having some mutation. The base sequence of the region encoding 16s rRNA (target nucleic acid) derived from such a mutant strain is almost the same as the base sequence reported in a typical wild strain, but there are many cases where some mutations exist. For this reason, amplification products prepared from clinically isolated strains and amplification products prepared from typical wild strains have a fluorescence intensity pattern observed at the spot of each DNA probe on the DNA microarray. Differences often occur.

  In particular, with regard to pneumococci, various resistant strains have been generated in clinical settings as a result of the application of treatment techniques that suppress the growth of antibiotics by administration of antibiotics. Under such circumstances, it is necessary to specify which of the various resistant strains, in addition to specifying the species of the causative bacteria present in the sample collected from a new patient. When it is necessary to identify this strain, not only the fluorescence intensity pattern observed at the spot of each DNA probe on the DNA microarray in the amplification product prepared from a typical wild strain, but also past cases The details of the fluorescence intensity patterns for amplification products prepared from various resistant strains observed are effective in comparison.

  The electronic medical record system according to the present invention is prepared from the causative bacteria present in a sample collected from a patient in a database of examination data included in the electronic medical records of many patients as past cases. In the amplified product, the fluorescence intensity pattern observed at the spot of each DNA probe on the DNA microarray is compared with the observed fluorescence intensity pattern in the sample collected from a new patient. It is possible to search for past cases with similar patterns. The doctor in charge of diagnosis and treatment of a new patient, for the past case with the most similar fluorescence intensity pattern, is described in the patient's electronic medical record, the diagnosis result, the reason for the diagnosis, the patient's symptoms, In addition, by referring to treatment records and the like given to the patient, it is greatly helpful in diagnosing a new patient in charge and determining a treatment policy.

  For example, the strain of the causal fungus infected by the new patient in charge is the strain of the causal fungus infected by the similar case patient selected by the above search, and the spot of each DNA probe on the DNA microarray If the pattern of fluorescence intensity observed at the point is similar, but it turns out that it shows a decisive difference, it can be estimated that there is a high concern that it is a new resistant strain that is not included in conventional cases. In that case, if it is necessary to carry out a more detailed inspection, it can be quickly determined.

In addition, when multiple causative organisms that are causing symptoms of a new patient are involved, just referring to the typical fluorescence intensity pattern observed in the infection of one causative organism is not appropriate. Diagnosis is difficult. At that time, if past cases where a plurality of causative bacteria are involved are found by searching for past cases with similar fluorescence intensity patterns, it will be a great reference for appropriate diagnosis.

A pattern of fluorescence intensity measured at a spot point of each DNA probe on a DNA microarray, which is measured in a patient, is expressed as, for example, a multidimensional vector having each element as the fluorescence intensity observed at each spot point. For example, regarding the DNA microarray shown in FIG. 6, A-1 to A-9, B-1 to B-7, C-1 to C-7, D-1 to D-6, E-1 to E-8. , F-1 to F-6, G-1 to G-7, H-1 to H-8, I-1 to I-7, J-1 to J-7. And a 72-dimensional vector {X ₁ ,..., X ₇₂ } having the fluorescence intensity (X _i ) observed at each spot point as an element.

A vector X _n {X _1n ,..., X _72n } corresponding to a new patient test result and a vector X _j {X _1j corresponding to a patient test result which is a past case stored in the electronic medical record system ,..., X _72j } and a past case (patient) showing a similar vector X _j {X _1j ,..., X _72j } is selected. For example, considering the difference between two vectors; ΔX _nj ≡X _n −X _j = {(X _1n −X _1j ),..., (X _72n −X _72j )}, similar vectors X _j {X _1j,. , X _72j } is determined to have a small size of ΔX _nj . That is, it can be determined that the smaller the magnitude of ΔX _nj ; {(X _1n −X _1j ) ² +... + (X _72n −X _72j ) ² } ^1/2 is similar. The magnitude of ΔX _nj for all of the vectors X _j {X _1j ,..., X _72j } corresponding to the test results of patients who are past cases stored in the electronic medical record system; {(X _1n −X _1j ) ² +... + (X _72n −X _72j ) ² } ^1/2 is calculated, and past cases (patients) are sorted in ascending order of ΔX _nj . That is, the magnitude of ΔX _nj corresponding to the Euclidean distance between two vectors; {(X _1n −X _1j ) ² +... + (X _72n −X _72j ) ² } ^1/2 This is a technique for performing the determination.

When the magnitude of ΔX _nj ; {(X _1n −X _1j ) ² +... + (X _72n −X _72j ) ² } ^1/2 is used as an index, Corresponding vector X _n {X _1n ,..., X _72n } and vector X _j {X _1j ,..., X _72j } corresponding to a test result of a past case (patient) have a relationship of X _n = kX _j Is satisfied, the magnitude of ΔX _nj is not determined to be zero. That is, the actual measurement result may have a relative fluctuation in fluorescence intensity due to a systematic error. In the actual measurement data, since there is no calibration means for such relative fluctuation, the uncalibrated measurement result is included as it is. In some cases, if relative intensity fluctuations have been calibrated, there is a possibility that past cases that are completely matched will not be searched as the most similar past case (patient).

Considering this point, the vector X _n {X _1n ,..., X _72n } and the vector X _j {X _1j ,..., X _72j } to be compared are normalized in advance and the difference between the two vectors; It is more preferable in many cases to consider ΔX _nj ≡X _n −X _j = {(X _1n −X _1j ),... (X _72n −X _72j )}.

Alternatively, the degree of similarity can be determined using an angle formed by two vectors as an index. Specifically, the inner product X _n · X _j ≡ {(X _1n · X _1j ) +... + (X _72n · X _72j )} and the size of the two vectors; X _n ≡ {(X _1n ) ² +... + (X _72n ) ² } ^1/2 , X _j ≡ {(X _1j ) ² +... + (X _72j ) ² } ^1/2 , X _n · X _j ≡ X _n · X _An angle θ formed by two vectors is defined using a relational expression of _j cos θ. In this method, it is determined that the degree of similarity between two vectors is higher as the angle θ formed by the directions of the two vectors is smaller. After normalization in advance, there is no substantial difference from the method that considers the difference between two vectors; ΔX _nj , but in principle, the angle θ formed by the direction of the two vectors is considered. The method is better.

Note that a scale other than the above-described index may be used as a scale of “distance” between two vectors.

As described above, the inspection data (primary data) can basically be converted into numerical data according to each inspection item. Therefore, regarding various inspection items, it is possible to obtain the similarity between data by integrating the examination results converted into numerical data, treating them as one vector, and obtaining the similarity between the vectors. .

  On the other hand, symptoms (clinical findings data) are not usually converted into numerical data, but can be converted into numerical data by scoring the presence / absence of each symptom and the degree of its severity. Alternatively, the description method of symptoms (clinical findings data) on the electronic medical record is described based on the preset criteria for scoring the presence or absence of each symptom and its severity. It is also possible. That is, by making the presence or absence of each symptom and the degree of severity thereof scored, it is possible to obtain the degree of similarity with respect to the symptom (clinical finding data) by the same method.

  For example, for a description of symptoms (clinical findings data) described in a normal sentence expression format, a part corresponding to a “keyword” indicating each symptom set in advance is applied by character search technology. After extraction, it is possible to score the presence or absence of each symptom and the degree of its severity. Specifically, a symptom comparison function can be implemented by incorporating a public text technology such as a full-text search system and Namazu (http://www.namazu.org/) into the system.

  Both examination data and symptoms (clinical findings data) reflect a patient's medical condition caused by a certain etiology, and each corresponds to a projection of the patient's medical condition onto different coordinate planes. For example, symptoms (clinical findings data) change over time as the patient progresses. Therefore, after considering the degree of progression of the disease state in a new patient, the total similarity is calculated by comprehensively considering the two aspects of examination data and symptoms (clinical findings data). For example, after calculating the similarity of the test data: ΔX and the similarity of the symptom: ΔY, weighting coefficients a and b are respectively added to calculate the total similarity as {aΔX + bΔY}. it can. For example, with respect to the weighting coefficient a for the similarity of examination data, when a = 0 is selected, it is used in a conventional electronic medical record system that searches for similar past cases based only on symptoms (clinical findings data). This corresponds to the search function. In general, the search function for past similar case patients used in the electronic medical record system of the present invention is configured to consider both the similarity of examination data: ΔX and the similarity of symptoms: ΔY at the same time. The weighting coefficient a for the data similarity is preferably selected to be at least as high as the weighting coefficient b for the symptom similarity: ΔY. For example, the ratio of the weighting coefficient a: weighting coefficient b is selected in a range of about a: b = 5: 1 to 1: 2, so that past similar case patients can be searched with more importance on the similarity of examination data. It becomes possible.

  The electronic medical record system according to the present invention is similar to a new patient among the past cases stored in the electronic medical record system when a doctor in charge makes a diagnosis regarding the etiology of the new patient, for example. By searching for similar case patients showing the symptom and the test result, referring also to the symptom and test result of the similar case patient, and proceeding with the diagnosis, it can be suitably used when making a diagnosis with higher accuracy.

Overall configuration of electronic medical record system according to the present invention, new patient test data, clinical findings data, patient test data corresponding to past cases, and clinical findings data provided in the electronic medical record system Based on the comparison with the above, the mechanism to search the test data and clinical findings data of patients (past cases), which shows extremely high similarity between the test data shown by new patients and the clinical findings data, the searched patients (past It is a figure which shows typically the outline | summary of the search function of a similar case which displays the test data of a case), clinical finding data, and treatment data, after removing the patient's personal information beforehand. In the electronic medical record system according to the present invention, recording and storage of individual patient's electronic medical records (examination data, clinical findings data, and treatment data), and of patients (past cases) stored in the form of electronic medical records It is a block diagram which shows typically the structure of the information processing apparatus utilized for achievement of the function which searches a similar case from an electronic medical record. In the electronic medical record system according to the present invention, recording and storage of individual patient's electronic medical records (examination data, clinical findings data, and treatment data), and of patients (past cases) stored in the form of electronic medical records It is a figure which shows typically the system configuration constructed | assembled in a client server type | mold about the information processing apparatus utilized for achievement of the function which searches a similar case from an electronic medical record. Using a DNA microarray in which multiple types of DNA probes are immobilized in an array, nucleic acid molecules contained in a sample are detected using a probe hybridization method. FIG. It is a figure which shows typically the procedure of the method of identifying the causative microbe of an infectious disease using the DNA probe fix | immobilized on the DNA microarray, and the principle. In the electronic medical record system according to the present invention, the results of the probe hybridization reaction employed as test data, in particular, DNAs that are respectively observed when the causative bacteria of the infectious disease are identified as S. aureus or E. coli It is a figure which shows an example of the two-dimensional image showing the fluorescence intensity in each spot point on a microarray.

Explanation of symbols

101 Patient data input process 102 Experimental measurement data database 103 Clinical data database 104 Experimental measurement data 105 Data matching process 106 Patient pointer of similar patient 107 Reference process of clinical data of similar patient 108 Personal information of similar patient by anonymization filter Removal process 109 Similar patient clinical data display process 201 External storage device 202 Central processing unit (CPU)
203 Internal Memory 204 Input / Output Edge 401 Sample 402 Biochemical Amplification Process 403 Label Mixing Process 404 DNA Microarray Construction Process 405 Hybridization Reaction Process 406 Fluorescence Measurement Process 407 Scan Image Process

Claims (7)

A patient electronic medical record database that stores patient examination data, clinical findings data, and treatment data as electronic medical record information for each patient;
An input device for inputting examination data, clinical findings data, and treatment data as new patient chart information;
Compare the test data and clinical findings data of the new patient input as medical record information with the patient test data and clinical findings data in the past cases stored in the patient electronic medical record database, A similar case patient selection mechanism for selecting one or more patients of past cases having the test data, clinical findings data, similar test data, and clinical findings data of the new patient,
A similar case patient chart information disclosure mechanism for electronically disclosing the contents of the selected similar case patient chart information to the viewer in a viewable form by the similar case patient selection mechanism. An electronic medical record system characterized by The selection mechanism of the similar case patient is:
One or more selected similar case patients are provided with a ranking function based on similar heights using as an index the degree of similarity with the test data and clinical findings data of the new patient. Item 3. The electronic medical record system according to Item 1. The medical record information disclosure mechanism for patients with similar cases
Apply non-viewable processing to personal information that is not used for diagnosis and treatment of patients with similar cases and is used only for patient identification, which is included in the medical record information of patients with similar cases disclosed The electronic medical record system according to claim 1, wherein the electronic medical record system has a function. The patient electronic medical record database is
4. The detailed contents of the performed examination and the measurement result are included as a part of the examination measurement result database included in the patient examination data constituting the database. An electronic medical record system according to claim 1. The details of the tests performed and the measurement results included as part of the test measurement results database included in the patient test data are:
5. The electronic medical record system according to claim 1, wherein the electronic medical record system includes electronic data indicating a result of a probe hybridization reaction for a plurality of DNA probes using a DNA microarray. As digitized data showing the results of probe hybridization reactions for a plurality of DNA probes using the DNA microarray,
When the result of the probe hybridization reaction for a plurality of DNA probes to identify the causative bacteria of the infection is indicated by the signal intensity derived from the label,
6. The electron according to claim 5, comprising scan image data of signal intensity derived from a label including a spot region of a plurality of DNA probes, or signal intensity data derived from a label at each spot point of a plurality of DNA probes. Medical chart system. The details of the tests performed and the measurement results included as part of the test measurement results database included in the patient test data are:
7. The computerized data according to claim 5 or 6, comprising digitized data indicating a result of a probe hybridization reaction for a plurality of DNA probes for identifying a causative agent of an infectious disease using a DNA microarray. Electronic medical record system.

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